Detection and characterization of a novel hepacivirus in long-tailed ground squirrels (Spermophilus undulatus) in China.

Rodent populations are known to be reservoirs of viruses with the potential to infect humans. However, a large number of such viruses remain undiscovered. In this study, we investigated the shedding of unknown viruses in long-tailed ground squirrel (Spermophilus undulatus) feces by high-throughput sequencing. A novel and highly divergent virus related to members of the genus Hepacivirus was identified in ground squirrel liver. This virus, tentatively named RHV-GS2015, was found to have a genome organization that is typical of hepaciviruses, including a long open reading frame encoding a polyprotein of 2763 aa. Sequence alignment of RHV-GS2015 with the most closely related hepaciviruses yielded p-distances of the NS3 and NS5B regions of 0.546 and 0.476, respectively, supporting the conclusion that RHV-GS2015 is a member of a new hepacivirus species, which we propose to be named "Hepacivirus P". Phylogenetic analysis of the NS3 and NS5B regions indicated that RHV-GS2015 shares common ancestry with other rodent hepaciviruses (species Hepacivirus E, and species Hepacivirus F), Norway rat hepacivirus 1 (species Hepacivirus G), and Norway rat hepacivirus 2 (species Hepacivirus H). A phylogenetic tree including the seven previously identified rodent hepaciviruses revealed extreme genetic heterogeneity among these viruses. RHV-GS2015 was detected in 7 out of 12 ground squirrel pools and was present in liver, lung, and spleen tissues. Furthermore, livers showed extremely high viral loads of RHV-GS2015, ranging from 2.5 × 106 to 2.0 × 108 copies/g. It is reasonable to assume that this novel virus is hepatotropic, like hepatitis C virus. The discovery of RHV-GS2015 extends our knowledge of the genetic diversity and host range of hepaciviruses, helping to elucidate their origins and evolution.

A B3LYP and MP2(full) theoretical investigation on the cooperativity effect between hydrogen-bonding and cation-molecule interactions and thermodynamic property in the 1: 2 (Na⁺: N-(Hydroxymethyl)acetamide) ternary complex.

The cooperativity effects between the O/N-H∙∙∙O hydrogen-bonding and Na⁺∙∙∙O cation-molecule interactions in the 1: 2 (Na⁺: N-(Hydroxymethyl)acetamide) systems were investigated at the B3LYP/6-311++G**, MP2(full)/6-311++G** and MP2(full)/aug-cc-pvtz levels. The thermodynamic cooperativity calculations were also carried out for two pathways of the ternary-complex formation. The result shows that, in most ternary complexes, the O/N-H∙∙∙O and Na⁺∙∙∙O interactions are weakened in comparison with those in binary systems, leading to the anti-cooperativity effects, in particular in the complexes in which only the Na⁺∙∙∙O interactions exist. Shifts of electron density confirm the existence of anti-cooperativity. The increase of favorable enthalpic contribution leads to the positive cooperativity effect with negative ΔG(coop.) on forming the ternary complex by initial N-(Hydroxymethyl)acetamide dimer followed by addition of Na⁺. In forming the ternary complex by Na⁺∙∙∙N-(Hydroxymethyl)acetamide with the second N-(Hydroxymethyl)acetamide unit, the large unfavorable entropy change leads to the negative cooperativity effect with positive ΔG(coop.). The ternary complex is more easily formed by the pathway in which Na⁺ binds to N-(Hydroxymethyl)acetamide dimer.

[Community structure of Great Gerbils (Rhombomys opimus) parasitic fleas in Junggar Basin focus and related epizootiological characteristics on plague].

OBJECTIVE: To understand the community structure of Rhombomys opimus parasitic fleas in the natural plague focus in Junggar Basin, so as to explain the relationship between the community structure and the prevalence of animal plague in this area. METHODS: The body fleas of R.opimus which were collected in 2005-2010 from the Junggar Basin was analysed by community ecology method, an each clustered flea community was compared with information of pathogenic and serological indicators of animal plague epidemic by statistic method. RESULTS: The community structure of R. opimus parasitic fleas included 19 species and was very complicated, with only 3 were identified as 'common' species but the others belonged to 'rare' species. Both the average richness and diversity of flea community were higher, as 1.66 and 1.5556, with dominance not obvious (0.332) and the homogeneity as moderate (0.5283). The community structure of R. opimus parasitic fleas could be changed by the following factors as: the rates of flea parasitic/flea index and the proportions of main fleas etc. The flea community could be divided into 5 clusters according to the above said elements. All these data showed that the fleas of R. opimus parasitic fleas possess the nature of multi-parasitism. CONCLUSIONS: The community of R. opimus parasitic fleas existed heterogeneous of geographic region, and this phenomenon associated with the animal plague epidemic strength.

Serial expression of the truncated fragments of the nucleocapsid protein of CCHFV and identification of the epitope region.

The Crimean-congo hemorrhagic fever virus (CCHFV) is a geographically widespread fatal pathogen. Identification of the epitope regions of the virus is important for the diagnosis and epidemiological studies of CCHFV infections. In this study, expression vectors carrying series truncated fragments of the NP (nucleocapsid protein) gene from the S fragment of CCHFV strain YL04057 were constructed. The recombinant proteins were expressed in E.coli and purified for detection. The antigenic of the truncated fragments of NP was detected with a polyclonal serum (rabbit) and 2 monoclonal (mAbs) (14B7 and 43E5) against CCHFV by Western-blot analyses. The results showed that the three expressed constructs, which all contained the region 235AA to 305AA could be detected by mAbs polyclonal serum. The results suggest that region 235-305 aa of NP is a highly antigenic region and is highly conserved in the NP protein.

[Study on the situation of plague in Junggar Basin of China].

OBJECTIVE: To understand the distribution, fauna, population structure of host animals and their parasitic fleas as well as popular dynamic of animal plague of natural plague foci in Junggar Basin. METHODS: Sample materials and data of animals and vector insects were collected using ecological methods and the population structures were analyzed statistically. F1 antibody of Yersinia pestis in rodents' serum and organ suspension was detected by means of IHA while the pathogen of Y. pestis in rodents and vector insects was detected by means of aetiological detections and the isolated Y. pestis was detected using biochemical methods. RESULTS: The small mammals which were found in Junggar Basin belonged to 17 species of 11 genera 7 families. Of them, 13 species of rodents were included whose parasitic fleas belonged to 19 species of 10 genera 8 families. The average coverage of Rhombomys opimus hole-community was 22.5% in Junggar Basin with the average density of R. opimus hole-community was 15.9/hm2 and the average rate of habitat of the hole-community was 70.2%. In the R. opimus community, the average density of rodents was 3.1/hole-community, and 34.4/hm2 in the nature plague foci. In the population structure of the hole-community of R. opimus, R. opimus accounted for 72.9% in the total captured rodents, Meriones meridianus was 24.5% while the others were 2.6%. In the nocturnal community of rodents, M. meridianus accounted for 64.0% in total captured rodents, Dipus sagitta was 15.1%, M. erythrourns was 7.5% and the others were 13.4%. In the rodents community of Junggar Basin, the rate of R. opimus with fleas was 84.9%, which was the highest, followed by M. tamariscinus, Euchoreutes naso and M. erythrourns, with the rates as 71.4%, 66.7% and 62.7% respectively. The rate of M. meridianus with fleas was 38.3%. There were 16 species of parasitic fleas in R. opimus, with the total flea index as 8.58 and the dominant species was Xenopsylla skrjabini. There were 17 and 16 kinds of fleas in M. erythrourns and M. meridianus respectively with the total flea index were 1.59 and 1.15, with dominant fleas were Nosopsyllus laeviceps and X. skrjabini. The serum and organ suspension of 3179 rodents which belonged to 12 species were detected by means of IHA, of them 174 samples were positive and the positive rate was 5.5%. There were 1356 samples of R. opimus in these materials, and 164 were positive, accounted for 12.1%. The samples of M. meridianus were 1255, with 9 positive, accounted for 0.7%. The samples of D. sagitta were 116 with 1 positive and the rate was 0.9%. The samples of other rodents were 452 but were all negative. There were in total 2975 organs collected from rodents, when detected by methods of isolated of Y. pestis. 15 strains of Y. pestis were isolated from 1243 R. opimus, and 2 strains isolated from 1230 M. meridianus. A total number of 11 647 fleas from rodents were detected by methods of isolated of Y. pestis in which 1 strain of Y. pestis was isolated from 4713 X. skrjabini, and 6 were isolated from 2101 Xenopsylla minax, 1 from 328 Xenopsylla conformis conformis and 1 from 250 Echidnophaga oschanini. Among the other 4255 fleas, none was isolated. The biochemical properties of these Y. pestis which isolated from Junggar Basin were positive of Maltose, Ejiao sugar and Glycerol, and negative of Rhamnose and Nitrogen, which were all strongly poisonous to mouse. CONCLUSION: The natural plague foci in Junggar Basin spread all over the whole Junggar Basin. There were animal plague cases found in 12 counties (cites) while Karamy, Bole, Jimusaer and Qitai were confirmed as plague foci counties (cities). Animals and vector insects of the foci were complicated but the ecological system was stable. R. opimus was recognized as the dominant host animal and its biochemical type belonged to the Middle Ages, suggesting that the foci was a new type of natural plague foci.

[Molecular analysis on the S gene of three Crimean-Congo hemorrhagic fever virus strains in China].

OBJECTIVE: To compare the molecular characteristics of 3 Crimean-Congo hemorrhagic fever viruses(CCHFV) isolated in Xinjiang province. METHODS: YL05035, YT05099 and LT05146 were isolated in 2005 from Hyalomma ticks and viral RNA was extracted from suckling mouse brains infected with these three strains respectively. The polymerase chain reaction(PCR) products of S segments from the 3 strains of CCHFV were directly sequenced. RESULTS: The full-length'S RNA from the 3 strains of CCHFV all comprised 1673 nucleotides with ORF of them including 1449 nucleotides and encoding a protein which comprised 482 amino acids in a viral complementary sense. The sequences indicated that the three strains of CCHFV isolated from ticks in Xinjiang province were highly homologenic. Data from the phylogenetic analysis showed that the obtained sequences were identical. The homology between 3 strains of CCHFV was 99.5%. Their homologies compared with that of the other strains isolated from other region of Xinjiang were also high at nucleotide levels (92.7%-99.8%). The three strains which were clustered together with 7001 strain and 79121 strain (isolated from patient and rat in Xinjiang respectively) was only different by 2%-3%. The genetic difference from the prototype CCHFV Nigerian strain IBAR10200 was 13%. In comparison, the Nigerian CCHFV tick isolate was more divergent when compared with the reference China strains 66019 and with the three variants mentioned above. CONCLUSION: The CCHFV isolated from China comprised a group of genetically high conserved strains.

[Geography and host distribution of Crimean-Congo hemorrhagic fever in the Tarim Basin].

OBJECTIVE: To determine the infective status and natural distribution of Xinjiang hemorrhagic fever (XHF; Crimean-Congo hemorrhagic fever, CCHF) in ticks, rodents and livestock in the Tarim Basin. METHODS: The pathogenic materials of ticks or rodents' viscera and blood samples of sheep were inoculated into sucking mouse of 24 to 48-hour old. Materials with typical clinic symptoms were identified with RPHA and IFA. RT-PCR was taken to detect special S gene segment of Crimean-Congo hemorrhagic fever virus (CCHFV) in the objective material. RESULTS: All the samples of ticks, rodents' viscera and blood samples of sheep from 21 counties (cities) in the Tarim Basin were divided into 422 groups and inoculated into sucking mouse at laboratory. 49 materials with typical clinic symptoms were obtained. The morbidity rate with typical clinic XHF was high in Bachu, Yuli, Yutian and Ruoqiang. There were 43 samples identified with RPHA with 6 positive samples and positive rate of 1.4%. The materials with positive RPHA were found in Yuli, Luntai and Yutian. 42 samples were identified with IFA and 13 positive samples with the positive rate of 3.1%. The positive materials of IFA were found in Bachu, Yuli, Minfeng, Luntai and Yutian. 32 samples were detected with RT-PCR and there were 31 samples with special S gene segment of CCHFV (329- 548 nt). The positive materials was widely distributed in Aksu, Awat, Bachu, Luopu, Yuli, Minfeng, Qiemo, Ruoqiang, Luntai and Yutian. The highest infective rate was in Hyalomma asiaticum kozlovi, and followed by sheep. S gene segment was detected in viscera of M. meridianus. CONCLUSION: XHF relied on the river in the southern part of Xinjiang and distributed in the areas with Populus euphratica shrub in desert and oasis in the Tarim Basin. The main vector and host were Hyalomma asiaticum kozlovi. Livestock such as sheep, camel, L. yarkandensis, M. meridianus and Euchoreutes naso could serve as the deposited host of XHF.

[Classification and diversity of tick community in Tarim basin].

OBJECTIVE: To investigate the distribution pattern and structural characteristics of tick community and to understand the diversity of the communities in Tarim Basin. METHODS: According to the geographical division and habitat types, survey sites were selected, and tick samples were collected and their species were identified. With the methods of community ecology, the richness, diversity and evenness of the tick community were calculated. The communities were classified by way of clustering analysis in combination with the environmental index of geology and vegetation. RESULTS: Totally 10 species belonging 5 genera of ticks were collected in the Basin. Hyalomma asiaticum asiaticum and Hyalomma asiaticum kozlovi were the dominant species in the area. The tick community was divided into 7 types in accordance with the environmental geology, vegetations, and their richness and coverage degree. CONCLUSIONS: There are abundant tick communities in the area of Tarim Basin, and a gradient change of the communities is continued in the ecological amplitude of this area.